The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the β-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1's processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in vivo.

Nuclear receptor-related 1 protein (Nurr1/NR4A2) is an orphan nuclear receptor (NR) that is considered to function without a canonical ligand-binding pocket (LBP). A crystal structure of the Nurr1 ligand-binding domain (LBD) revealed no physical space in the conserved region where other NRs with solvent accessible apo-protein LBPs bind synthetic and natural ligands. Using solution nuclear magnetic resonance spectroscopy, hydrogen/deuterium exchange mass spectrometry, and molecular dynamics simulations, we show that the putative canonical Nurr1 LBP is dynamic with high solvent accessibility, exchanges between two or more conformations on the microsecond-to-millisecond timescale, and can expand from the collapsed crystallized conformation to allow binding of unsaturated fatty acids. These findings should stimulate future studies to probe the ligandability and druggability of Nurr1 for both endogenous and synthetic ligands, which could lead to new therapeutics for Nurr1-related diseases, including Parkinson's disease and schizophrenia.

Terminal uridyltransferases (TUTases) execute 3' RNA uridylation across protists, fungi, metazoan and plant species. Uridylation plays a particularly prominent role in RNA processing pathways of kinetoplastid protists typified by the causative agent of African sleeping sickness, Trypanosoma brucei In mitochondria of this pathogen, most mRNAs are internally modified by U-insertion/deletion editing while guide RNAs and rRNAs are U-tailed. The founding member of TUTase family, RNA editing TUTase 1 (RET1), functions as a subunit of the 3' processome in uridylation of gRNA precursors and mature guide RNAs. Along with KPAP1 poly(A) polymerase, RET1 also participates in mRNA translational activation. RET1 is divergent from human TUTases and is essential for parasite viability in the mammalian host and the insect vector. Given its robust in vitro activity, RET1 represents an attractive target for trypanocide development. Here, we report high-resolution crystal structures of the RET1 catalytic core alone and in complex with UTP analogs. These structures reveal a tight docking of the conserved nucleotidyl transferase bi-domain module with a RET1-specific C2H2 zinc finger and RNA recognition (RRM) domains. Furthermore, we define RET1 region required for incorporation into the 3' processome, determinants for RNA binding, subunit oligomerization and processive UTP incorporation, and predict druggable pockets.

Nuclear receptor (NR) transcription factors bind various coreceptors, small-molecule ligands, DNA response element sequences, and transcriptional coregulator proteins to affect gene transcription. Small-molecule ligands and DNA are known to influence receptor structure, coregulator protein interaction, and function; however, little is known on the mechanism of synergy between ligand and DNA. Using quantitative biochemical, biophysical, and solution structural methods, including 13C-detected nuclear magnetic resonance and hydrogen/deuterium exchange (HDX) mass spectrometry, we show that ligand and DNA cooperatively recruit the intrinsically disordered steroid receptor coactivator-2 (SRC-2/TIF2/GRIP1/NCoA-2) receptor interaction domain to peroxisome proliferator-activated receptor gamma-retinoid X receptor alpha (PPARγ-RXRα) heterodimer and reveal the binding determinants of the complex. Our data reveal a thermodynamic mechanism by which DNA binding propagates a conformational change in PPARγ-RXRα, stabilizes the receptor ligand binding domain dimer interface, and impacts ligand potency and cooperativity in NR coactivator recruitment.

IGF2BP2 (IMP2) is an RNA-binding protein that contributes to cancer tumorigenesis and metabolic disorders. Structural studies focused on individual IMP2 domains have provided important mechanistic insights into IMP2 function; however, structural information on full-length IMP2 is lacking but necessary to understand how to target IMP2 activity in drug discovery. In this study, we investigated the behavior of full-length IMP2 and the influence of RNA binding using biophysical and structural methods including mass photometry, hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), and small angle x-ray scattering (SAXS). We found that full-length IMP2 forms multiple oligomeric states but predominantly adopts a dimeric conformation. Molecular models derived from SAXS data suggest the dimer is formed in a head-to-tail orientation by the KH34 and RRM1 domains. Upon RNA binding, IMP2 forms a pseudo-symmetric dimer different from its apo/RNA-free state, with the KH12 domains of each IMP2 molecule forming the dimer interface. We also found that the formation of IMP2 oligomeric species, which includes dimers and higher-order oligomers, is sensitive to ionic strength and RNA binding. Our findings provide the first insight into the structural properties of full-length IMP2, which may lead to novel opportunities for disrupting its function with more effective IMP2 inhibitors.

Muscle cell fusion is a multistep process where the final step of the reaction drives progression beyond early hemifusion events to complete fusion. This step requires activity of the muscle-specific fusogen Myomerger, a single-pass transmembrane protein containing 84 amino acids with an ectodomain that includes two α-helices. Previous studies have demonstrated that Myomerger acts by destabilizing membranes through generation of elastic stresses in the outer leaflet of the plasma membrane. An obvious question is how such destabilizing activity might be regulated to avoid membrane and cellular damage, and how the two juxtaposed helices cooperate in fusion. Using cellular fusion assays and in vitro liposome assays, we report that the two helices possess unique characteristics, both of which are needed for full activity of the protein. We demonstrate that externalized phosphatidylserine (PS), a lipid previously implicated in myoblast fusion, has a determinant role in the regulation of Myomerger activity. The membrane-proximal, amphipathic Helix-1 is normally disordered and its α-helical structure is induced by PS, making membrane interactions more efficacious. The distal, more hydrophobic Helix-2 is intrinsically ordered, possesses an ability to insert into membranes, and augments the membrane-stressing effects of Helix-1. These data reveal that Myomerger fusogenic activity is an exquisitely orchestrated event involving its two ectodomain helices, which are controlled by membrane lipid composition, providing an explanation as to how its membrane-stressing activity is spatially and temporally regulated during the final step of myoblast fusion.

Crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) have revealed overlapping binding modes for synthetic and natural/endogenous ligands, indicating competition for the orthosteric pocket. Here we show that cobinding of a synthetic ligand to the orthosteric pocket can push natural and endogenous PPARγ ligands (fatty acids) out of the orthosteric pocket towards an alternate ligand-binding site near the functionally important omega (Ω)-loop. X-ray crystallography, NMR spectroscopy, all-atom molecular dynamics simulations, and mutagenesis coupled to quantitative biochemical functional and cellular assays reveal that synthetic ligand and fatty acid cobinding can form a 'ligand link' to the Ω-loop and synergistically affect the structure and function of PPARγ. These findings contribute to a growing body of evidence indicating ligand binding to nuclear receptors can be more complex than the classical one-for-one orthosteric exchange of a natural or endogenous ligand with a synthetic ligand.

Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERBβ ligand-binding domain (LBD). We solved two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

GPR88 is a neuronal cerebral orphan G-protein-coupled receptor (GPCR) that has been linked to various psychiatric disorders. However, no extensive description of its localization has been provided so far. Here, we investigate the spatiotemporal expression of the GPR88 in prenatal and postnatal rat tissues by using in situ hybridization and immunohistochemistry. GPR88 protein was initially detected at embryonic day 16 (E16) in the striatal primordium. From E16-E20 to adulthood, the highest expression levels of both protein and mRNA were observed in striatum, olfactory tubercle, nucleus accumbens, amygdala, and neocortex, whereas in spinal cord, pons, and medulla GPR88 expression remains discrete. We observed an intracellular redistribution of GPR88 during cortical lamination. In the cortical plate of the developing cortex, GPR88 presents a classical GPCR plasma membrane/cytoplasmic localization that shifts, on the day of birth, to nuclei of neurons progressively settling in layers V to II. This intranuclear localization remains throughout adulthood and was also detected in monkey and human cortex as well as in the amygdala and hypothalamus of rats. Apart from the central nervous system, GPR88 was transiently expressed at high levels in peripheral tissues, including adrenal cortex (E16-E21) and cochlear ganglia (E19-P3), and also at moderate levels in retina (E18-E19) and spleen (E21-P7). The description of the GPR88 anatomical expression pattern may provide precious functional insights into this novel receptor. Furthermore, the GRP88 nuclear localization suggests nonclassical GPCR modes of action of the protein that could be relevant for cortical development and psychiatric disorders. J. Comp. Neurol. 524:2776-2802, 2016. © 2016 Wiley Periodicals, Inc.